Therapeutic composition comprising an antigen or an in vivo generator of a compound comprising an amino acid sequence

ABSTRACT

A therapeutic composition comprising (i) at least one antigen or at least one in vivo generator of a compound comprising an amino acid sequence and (ii) at least one adjuvant comprising at least one pharmaceutically acceptable and water-soluble salt of an organic anion and a metal cation.

BACKGROUND OF THE INVENTION

(i) Field of the Invention

The present invention relates to a therapeutic composition comprising atleast one antigen, in particular an antigen of viral, bacterial orparasitic origin, or at least one in vivo generator of a compoundcomprising an amino acid sequence, and at least one adjuvant.

(ii) Description of Related Art

The use of adjuvants in therapeutic compositions of the vaccine type hasbeen known for a long time. The main objective of these adjuvants is toallow an increase in the immune response. These adjuvants are diverse innature. They may, for example, consist of liposomes, oily phases, forexample the Freund type of adjuvants, generally used in the form of anemulsion with an aqueous phase, or, more commonly, may consist ofwater-insoluble inorganic salts. These inorganic salts may consist, forexample, of aluminum hydroxide, zinc sulfate, colloidal iron hydroxide,calcium phosphate or calcium chloride. Aluminum hydroxide (Al(OH)₃) isthe most commonly used adjuvant. These adjuvants are described inparticular in the article by Rajesh K. Gupta et al “Adjuvants, balancebetween toxicity and adjuvanticity”, Vaccine, Vol. 11, issue 3, 1993,pages 993-306.

The adjuvants mentioned above have the drawback of limited efficiency.Moreover, they may induce a certain toxicity with regard to individualstreated. More particularly, when these therapeutic compositions areinjected, the formation of lesions and other local reactions such asgranulomae is observed at the point of injection. These drawbacks areless pronounced when the adjuvant in aluminum hydroxide. Accordingly,the latter compound is one of the most commonly used adjuvants.Recently, however, aluminum hydroxide, like all aluminum-basedcompounds, has come to be suspected of being a factor promoting theappearance of certain diseases, such as renal dysfunctions orAlzheimer's disease. In addition, it is known that aluminum hydroxideefficiently induces only humoral immunity and not cell immunity.

SUMMARY AND OBJECTS OF THE INVENTION

A first object of the invention is to provide therapeutic compositionscomprising an adjuvant which allows an increase in the immune responsewhich is at least equal to that imparted by aluminum hydroxide, withoutcausing lesions or local reactions of the granuloma type and which isnot liable to promote the appearance of diseases in the individualtreated.

Another object of the invention is to provide a therapeutic compositioncomprising an adjuvant which efficiently induces both cell immunity andhumoral immunity.

Yet another object of the invention is to provide a method for making atherapeutic composition using the adjuvant of the invention.

In a first aspect, the present invention relates to a therapeuticcomposition comprising (i) at least one antigen or at least one in vivogenerator of a compound comprising an amino acid sequence and (ii) atleast one adjuvant comprising at least one pharmaceutically acceptableand water-soluble salt of an organic anion and a metal cation.

In a second aspect, the present invention relates to a method of makinga composition intended for the prevention or treatment of infectiousdiseases comprising combining (a) at least one adjuvant comprising atleast one pharmaceutically acceptable and water-soluble salt of anorganic anion and a metal cation and (b) at least one antigen or atleast one in vivo generator of a compound comprising an amino acidsequence.

In a third aspect, the present invention relates to a method fortreating a functional disease comprising administering to a patient thetherapeutic composition as described above.

In another aspect, the invention relates to an adjuvant compositioncomprising a pharmaceutically acceptable and water-soluble salt and (a)an oily adjuvant, (b) a surfactant and/or (c) an oily adjuvant combinedwith a surfactant.

With the foregoing as well as other objects advantages and features ofthe invention that will become hereinafter apparent, the nature of theinvention may be better understood by reference to the followingdetailed description of the preferred embodiments and to the appendedclaims.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the sense of the present invention, a water-soluble salt may be suchthat its solubility in water is greater than or equal to 10 g/l,preferably between 10 and 2000 g/l.

The metal cation constituting said pharmaceutically acceptable salt ispreferably a divalent cation. This cation is advantageously a cation ofa metal chosen from the group consisting of manganese, calcium and zinc.Manganese is a very particularly preferred metal within the context ofthe present invention. The reason for this is that it has been observedthat pharmaceutically acceptable and water-soluble salts according tothe invention comprising an Mn²⁺ cation allow the induction of aparticularly large immune response, while at the same time being low intoxicity.

The organic anion constituting said pharmaceutically acceptable salt isadvantageously an anion of a compound comprising at least one oxygenatedfunctional group, preferably a phosphoric group —PO₄H₂, or a carboxylicgroup —COOH.

Glycerophosphoric acid is a preferred anion containing a phosphoricgroup.

The preferred anions comprising at least one carboxylic group arederived from compounds chosen from:

acid saccharides, preferably acid saccharides having from 5 to 7 carbonatoms, more preferably those having 6 or 7 carbon atoms,

mono- or polycarboxylic acids,

amino acids.

The preferred mono- or polycarboxylic acids are fumaric acid and thecompounds of the formula (I):

where:

R represents COOH, CH₃CO, CH₃ or CH₂OH,

R′ represents H or COOH and

s, t and u, which may be identical or different, are between 0 and 3.

Preferred compounds of formula (I) are acetic acid, lactic acid,tartaric acid, malic acid, citric acid and pyruvic acid.

In the sense of the present invention, an acid saccharide is of asaccharide comprising at least one carboxylic function, a saccharidebeing a glucide consisting of reducing sugars. These acid saccharidesare advantageously aldose derivatives obtained by oxidation of theprimary alcohol function or of the aldehyde function into a carboxylicfunction. Such compounds may most particularly include of gluconic acid,glucuronic acid, fructoheptonic acid, gluconoheptonic acid andglucoheptonic acid. When the organic anion is derived from an aminoacid, this amino acid may be an α-amino acid such as glutamic acid,methionine and, most particularly, aspartic acid.

A pharmaceutically acceptable, water-soluble salt according to theinvention may comprise a polyvalent cation, especially a divalentcation, combined with an organic anion or with several organic anions ofdifferent nature. Thus, by way of example, a divalent cation such as thecalcium cation may be combined with an anion derived fromgluconoheptonic acid and an anion derived from gluconic acid.

A water-soluble adjuvant most particularly preferred within the contextof the present invention consists of manganese gluconate.

A therapeutic composition according to the present invention maycomprise between 0.01 and 1000 mg/ml, preferably between 0.1 and 150mg/ml, of an adjuvant as defined above.

A therapeutic composition according to the invention may be prepared bysimple mixing of an aqueous suspension containing the antigen or said invivo generator with an aqueous solution of the salt defined above.

Besides the adjuvant comprising a pharmaceutically acceptable andwater-soluble salt as defined above, the therapeutic compositionaccording to the invention may also comprise an oily adjuvant. In such acase, the therapeutic composition according to the invention isadvantageously in the form of an emulsion combining at least one aqueousphase and at least one oily phase.

This emulsion may be of the water-in-oil (W/O) type or, preferably, ofthe oil-in-water (O/W), water-in-oil-in-water (W/O/W) or microemulsiontype. Such an emulsion may be prepared according to the standard methodsfor the preparation of an emulsion, in particular according to theprocesses described in patent applications EP-A-489,181 andEP-A-481,982. Thus, the oil constituting the oily phase may beemulsified, with stirring, with the aqueous phase including an aqueoussolution or suspension containing the antigen.

An emulsion according to the invention may contain, by weight, from 0.5%to 99.5% of oily phase per 99.5% to 0.5% of aqueous phase, preferablyfrom 5% to 95% of oily phase per 95 to 5% of aqueous phase and, morepreferably, from 25 to 27% of oily phase per 75 to 25% of aqueous phase.The emulsion must be stable preferably for at least 12 months when it isstored at 4° C.

The oily adjuvant may be a mineral oil, a non-mineral oil or a mixtureof a mineral oil and a non-mineral oil. Said mineral oils may be naturalor synthetic. Said non-mineral oils may be of plant, animal or syntheticorigin. The non-mineral oils are advantageously metabolizable. All theseoils are devoid of toxic effects with regard to the host organism intowhich the composition of the invention is administered. They arepreferably liquid at the storage temperature (about +4° C.) or at leastmake it possible to give emulsions which are liquid at this temperature.An advantageous mineral oil according to the invention may include anoil comprising a linear carbon chain having a number of carbon atomspreferably greater than 16, and free of aromatic compounds. Such oilsmay, for example, be those marketed under the name “MARCOL 52” (producedby Esso France) or “DRAKEOL 6VR” (produced by Penreco USA).

Examples of synthetic non-mineral oils which may be mentioned arepolyisobutenes, polyisopropenes, esters of alcohols and fatty acids,such as, for example, ethyl oleate and isopropyl myristate, mono-, di-or triglycerides, propylene glycol esters, partial glycerides such ascorn oil glycerides, for instance those marketed by the company SEPPICunder the name LANOL ™, maisin and oleyl oleate. Among the plant oilswhich may be mentioned are unsaturated oils rich in oleic acid which arebiodegradable, for example groundnut oil, olive oil, sesame oil, soyaoil or wheatgerm oil.

The animal oils may include in particular squalene, squalane orspermaceti oil.

Moreover, when it is in the form of an emulsion as defined above, thetherapeutic composition according to the invention may alsoadvantageously contain one or more surface-active agents. The latteragent has a lipophilic or hydrophilic nature characterized by an HLB(hydrophilic-lipophilic balance) value between 1 and 19.

Such a surfactant may include:

* an alkylpolyglycoside or a mixture of alkyl-polyglycosides of formulaRa—(O)—Zn where Ra represents a linear or branched saturated aliphaticradical comprising from 4 to 24 carbon atoms, preferably from 8 to 22carbon atoms, Z is a sugar residue, preferably glucose, and n is between1 and 5, preferably between 1.1 and 2,

* saponins,

* lecithins,

* polyoxyethylated alkanols such an those marketed under the name BRIJby the company ICI,

* polymers comprising polyoxyethylene and polyoxypropylene blocks, suchas those marketed under the name PLURONIC by the company BASF.

Particularly preferred surfactants are polyethylene glycol estersobtained by condensation of a fatty acid, in particular a fatty acidwhich is liquid at 20° C., with a polyethylene glycol of molecularweight between 80 and 2000; such a surfactant is marketed by the companySEPPIC under the tradename SIMULSOL 2599.

Another surface-active agent preferred within the context of the presentinvention is an ester obtained by condensation of a fatty acid,advantageously a fatty acid which is liquid at 20° C., with a sugar,sorbitol, glycerol or a polyglycerol, preferably a polyglycerolcomprising from 2 to 5 glycerol units. Said sugar may be glucose,sucrose or, preferably, mannitol. By way of particularly preferredmannitol ester, there may be mentioned mannitol oleates obtained bydehydration of the polyhydroxylated carbon chain of mannitol whichundergoes 1-4 or 2-6 cyclization.

Derivatives of these sugar esters, of polyethylene glycol, of sorbitol,of glycerol or of polyglycerol may also be used. These derivatives havea hydrophilicity which is modified in particular by grafting hydrophilicfunctions such as alcohol, polyol, ethylene oxide, propylene oxide,carboxylic acid, amine or amide. Such derivatives may, for example,include polyoxyethylated fatty esters of sorbitan, such as the TWEENs(conf. International Cosmetic Ingredient Dictionary, 5th ed. 1993).

Another preferred type of surfactant is ethoxylated plant oils such as,for example, ethoxylated castor oil, this oil being optionallyhydrogenated.

A surface-active agent according to the invention is preferablypharmaceutically acceptable for an injectable use; it must in particularbe free of heavy metals and have very low acid numbers or peroxidenumbers. It is also desirable for it to satisfy the harmlessness teststandards such as, for example, those described by S. S. Berlin, Annalsof Allergy, 1962, 20, 473 or the tests of abnormal toxicity described inthe European Pharmacopoeia. The surface-active agent is preferablycombined with the oily adjuvant before formation of the emulsion.

The concentration of surface-active agent in the therapeutic compositionmay be between 0.01 and 500 mg/ml, preferably between 0.1 and 200 mg/ml.

Oils associated with a surface-active agent (mannitol ester) which aremost particularly suitable within the context of the present inventionare those marketed by the company SEPPIC under the tradename“MONTANIDE”. The nature of these oils, the type of emulsion which can beobtained with them and the properties (viscosity and conductivity) ofthese emulsions are featured in Table 1 below:

TABLE 1 Aque- ous phase/ Type emul- Oil/ of sion Vis- ConductivityCommercial manitol emul- (% by cosity at 25° C. No. name esters sionweight) (mPa s) (μS cm⁻¹) 1 MONTANIDE Mineral O/W 75% 20 5000 ISA 25 2MONTANIDE Mineral O/W 75% 20 5000 ISA 25A + avridine 3 MONTANIDE MineralO/W 75% 25 1000 ISA 28 + ethyl- oleate 4 MONTANIDE Mineral W/O/ 50% 501000 ISA 206 W 5 MONTANIDE Mineral W/O 50% 200 1 ISA 50 6 MONTANIDEPlant W/O 30% 70 1 ISA 708 *Avridine =N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine.

The oily adjuvant may also include a self-emulsifiable oil, that is tosay an oily preparation capable of forming a stable emulsion with anaqueous phase, with virtually no energy input, for example by dispersionin the aqueous phase by slow mechanical stirring. In this respect,self-emulsifiable oils such as those known in the European Pharmacopoeiaunder the names Labrafil and Simulsol may be mentioned. These oils arepolyglycolyzed glycerides.

Preferred self-emulsifiable oils are those described in French patentapplication No. 9500497 filed on 18 January 1995, in the name of theApplicant, entitled “Utilisation d'esters d'acides gras éthoxylés commecomposants auto-émulsionnables notamment utiles pour la préparation decompositions phytosanitaires ou de médicaments à usage vétérinaire ouhumain” [Use of ethoxylated fatty acid esters as self-emulsifiablecomponents which are particularly useful for the preparation ofplant-protection compositions or medicinal products for human orveterinary use], These oils include ethoxylated fatty acid esterscorresponding to one of the following formulae:

in which:

R₁ R₃, R₅, R₆, R₈ and R₁₀ represent a saturated or unsaturated, linearor branched hydrocarbon chain having from 5 to 30 carbon atoms;

R₂, R₄, R₇, and R₉ represent a saturated or unsaturated, linear orbranched hydrocarbon chain having from 1 to 5 carbon atoms;

the total number of ethylene oxide molecules represented in theabovementioned formulae II,III and IV by k, 1+m and n+p+q, respectively,being an integer such that the HLB value of said compounds is betweenabout 4 and about 10, preferably between about 5 and about 9.

R₁ is preferably chosen from palmitic, stearic, ricinoleic, oleic,linoleic and linolenic acid residues, R₂ represents a methyl radical andk is an integer between 1 and 5, preferably equal to 2, and moreover thepreferred ethoxylated fatty acid esters of formula III are those inwhich:

(i)—R₆, R₈ and R₁₀ represent hydrocarbon chains having from 16 to 22carbon atoms, corresponding in particular to the fatty chains ofrapeseed oil, of corn oil, of soya oil, of groundnut oil and of apricotkernel oil,

R₇ and R₉ represent a methylene group, CH₂;

n, p and q represent integers such that their sum is between 3 and 30,and preferably equal to 20; or

(ii)—R₆, R₈ and R₁₀ represent hydrocarbon chains corresponding to thefatty chains of castor oil;

R₇ and R₉ represent a methylene radical, CH₂;

n, p and q represent integers such that their sum is between 5 and 7.

The concentration of self-emulsifiable oil in the therapeuticcomposition according to the invention may be approximately between 5and 700 g/l, preferably approximately between 10 and 500 g/l.

Besides the oily phase and the aqueous phase, the composition accordingto the invention may contain a conventional immunostimulatory agent suchas Avridine®, i.e.N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)propanediamine, MDP (muramyldipeptide) derivatives, especially threonyl-MDP, mycolic acidderivatives or Lipid A derivatives.

The therapeutic composition according to the invention may also compriseone or more surface-active agents, in the absence of any oily adjuvant.

The therapeutic composition is then in the form of a micellar solution.This may be prepared by simple mixing of the surface-active agent with adispersion in water of the antigen or of the in vivo antigen generator.

The surface-active agent may be chosen from the surface-active agentsdescribed above, in combination with an oily adjuvant.

Said micellar solution may contain from 0.5 to 500 mg/ml, preferablyfrom 1 to 250 mg/l, of surface-active agent.

A therapeutic composition according to the present invention maycomprise an antigen such as a virus, a microorganism, more particularlya bacterium or parasite, or a compound comprising a peptide chain. Sucha compound may include a protein or a glycoprotein, especially a proteinor glycoprotein obtained from a microorganism, a synthetic peptide or aprotein or a peptide obtained by genetic engineering. Said virus andmicroorganism may be totally inactivated or live and attenuated. By wayof virus which may constitute an antigen according to the presentinvention, there may be mentioned the rabies virus, Aujeszky viruses,influenza viruses, the virus of foot-and-mouth disease or HIV viruses.By way of microorganism of bacterial type which may constitute anantigen according to the present invention, there may be mentioned E.coli and those of the genera Pasteurella, Furonculosis, Vibriosis,Staphylococcus and Streptococcus. By way of parasite, there may bementioned those of the genera Trypanosoma, Plasmodium and Leishmania.

A therapeutic composition according to the invention comprises anantigen concentration which depends upon the nature of this antigen andon the nature of the individual treated. It is, however, particularlynoteworthy that an adjuvant according to the invention, which may or maynot be combined with an oily adjuvant and/or a surface-active agent asare defined above, makes it possible to decrease in an appreciablemanner the usual dose of antigen required. The appropriate antigenconcentration may be determined conventionally by those skilled in theart. This dose is generally of the order of 0.1 μg/ml to 1 g/ml, moregenerally between 1 μg/ml and 100 mg/l.

A therapeutic composition according to the invention may also comprisean in vivo generator of a compound comprising an amino acid sequence,that is to say a biological compound capable of expressing such acompound in the host organism into which said in vivo generator has beenintroduced. The compound comprising the amino acid sequence may be aprotein, a peptide or a glycoprotein.

These in vivo generators are generally obtained by genetic engineeringprocesses.

More particularly, they may comprise living microorganisms, generally avirus, acting as a recombinant vector, into which is inserted anucleotide sequence, in particular an exogenous gene. Those compoundsare known as such and are used in particular as recombinant sub-unitvaccines.

In this regard, reference may be made to the article by M. Eloit et al.,Journal of virology (1990) 71, 2925-2431, to International ApplicationWO-A-91.00107 or to International Application WO-A-94/16681.

The microorganism constituting a recombinant sub-unit vaccine isadvantageously a non-sheathed recombinant virus chosen, for example,from adenoviruses, the virus of the vaccine, canarypox virus, herpesviruses and baculoviruses. The exogenous gene inserted into themicroorganism may, for example, be derived from an Aujeszky virus orHIV.

The in vivo generators according to the invention may also comprise arecombinant plasmid comprising an exogenous nucleotide sequence capableof expressing in a host organism a compound comprising an amino acidsequence. Such recombinant plasmids and their mode of administrationinto a host organism were described in 1990 by Lin et al., circulation82:2217,2221; Cox et al., J. of Virol., Sept. 1993, 67, 9, 5664-5667 andin International Application WO/FR 95/00345 dated Mar. 21, 1995, in thename of the Applicant, entitled “Une composition comprenant un plasmiderecombinant et ses utilisations comme vaccin et médicament” [Acomposition comprising a recombinant plasmid and uses thereof as vaccineand medicinal product].

Depending on the nature of the nucleotide sequence comprised within thein vivo generator, the compound comprising the amino acid sequence whichis expressed within the host organism may:

(i) be an antigen, and permit triggering of an immune reaction,

(ii) have a curative action with respect to a disease, essentially afunctional disease, which has been triggered in the host organism. Inthis case, the in vivo generator allows a therapeutic treatment of thehost, of the gene therapy type.

By way of example, such a curative action may comprise the in vivocytokine generator, such as the interleukins, in particular interleukin2. The latter permit triggering or reinforcement of an immune reactionaimed at the selective removal of cancer cells.

The concentration of said in vivo generator in the therapeuticcomposition according to the invention depends, here also, in particularon the nature of said generator and on the host into which it isadministered. This concentration may readily be determined by thoseskilled in the art, on the basis of routine experiment.

As a guide, it may, however, be pointed out that when the in vivogenerator a recombinant microorganism, its concentration in thetherapeutic composition according to the invention may be between 10²and 10¹⁵ microorganisms/ml, preferably between 10⁵ and 10¹²microorganisms/ml.

When the in vivo generator is a recombinant plasmid, its concentrationin the therapeutic composition according to the invention may be between0.01 and 100 g/l.

A therapeutic composition according to the invention may be used as apreventive or curative medicinal product. Depending on the nature of theantigen or of the in vivo generator, a therapeutic composition accordingto the invention may be administered to fish, to crustaceans such asshrimps, to poultry, in particular geese, turkeys, pigeons and chickens,to canines such as dogs, to felines such as cats, to pigs, primates,cattle, sheep and horses. The therapeutic composition according to theinvention comprising a pharmaceutically acceptable water-soluble salt asdefined above may also be administered to man. The therapeuticcomposition may be administered in a conventional manner, in particularby subcutaneous, intramuscular or intraperitoneal injection or via theoral or mucosal route.

Another aspect of the invention is use of an adjuvant comprising apharmaceutically acceptable water-soluble salt as defined above for thepreparation of a vaccine intended for the prevention or treatment of aninfectious disease, in particular an infectious disease generated by avirus or a microorganism such as those mentioned above.

Another aspect of the invention is the use of a pharmaceuticallyacceptable water-soluble salt for the preparation of a therapeuticcomposition intended to treat a functional disease, such as cancer ormucoviscidosis.

In one other of these uses, said pharmaceutically acceptable salt may becombined with at least one of: an oily adjuvant, a surface-active agentand one oily adjuvant which is itself combined with a surface-activeagent; these oily adjuvants and surfactants being as defined above.

Adjuvant compositions comprising said pharmaceutically acceptable saltand the oily adjuvant and/or the surfactants mentioned above constituteyet another aspect of the invention. Where appropriate, these adjuvantcompositions comprise at least one aqueous phase.

In the latter case, the adjuvant compositions according to theinvention, comprising at least one oily phase and, where appropriate, asurfactant, may be in the form of an emulsion. This emulsion may be ofthe W/O, O/W, W/O/N or microemulsion type.

These emulsions may comprise, by weight, from 0.5% to 99.5% of oilyphase per 99.5% to 0.5% of aqueous phase, preferably from 5 to 95% ofoily phase per 95 to 5% of aqueous phase and, more preferably, from 25to 75% of oily phase per 75 to 25% of aqueous phase.

Where appropriate, they may comprise from 0.01 to 500 mg/ml, preferablyfrom 0.1 to 200 mg/ml, of at least one surfactant.

When the adjuvant composition according to the invention comprises,besides the pharmaceutically acceptable salt and an aqueous phase, onlyone or more surfactants, it is then in the form of a micellar solution.The surfactant content of this micellar solution may be between 0.01 and900 mg/ml, preferably between 1 and 250 mg/mi.

An adjuvant composition according to the invention usually comprisesfrom 0.02 to 3000 mg/ml, preferably 0.1 to 1000 mg/ml and morepreferably from 0.1 to 150 mg/ml, of a pharmaceutically acceptable saltaccording to the invention.

These compositions are useful for preparing the therapeutic compositionsaccording to the invention.

The latter compositions may then be prepared by simple mixing of theadjuvant composition with a composition comprising an antigen or an invivo generator of a compound comprising an amino acid sequence.

The invention will be better understood with regard to the examples andfigures below.

The examples were performed on OF1 mice whose average weight was between18 and 20 g.

The results expressed are an average of the results obtained on 10 mice.

The therapeutic compositions comprised Sigma Grade V bovine serumalbumin (BSA) as antigen.

The therapeutic compositions were injected subcutaneously.

The humoral immune response was determined by assay of the total IgG andof the total IgG₁, according to the ELISA method.

The cell immune response was determined by assay of the IgG₂a, accordingto the ELISA method.

The antibody levels mentioned in the examples correspond to the lastdilution above the background noise.

The following abbreviations are used in the examples:

Glu=gluconate

Fruhp=fructoheptonate

Gly=glycerophosphate

Gluhp=glucoheptonate

Al(OH)₃=aluminum hydroxide

Asp=aspartic acid

Thus, by way of example, GluMn is manganese gluconate.

EXAMPLE 1

Therapeutic compositions (or doses) of 100 μl each, comprising 50 μg ofBSA, are prepared.

These therapeutic compositions were injected into the mice on D0 (i.e.the day of injection).

The level of anti-BSA antibodies was determined 14 and 28 days after theinjection (primary response).

On D28, the same therapeutic composition was injected into the samemice.

The level of anti-BSA antibodies was determined on D42 and D56.

The results obtained are featured in Table II below.

TABLE II anti- BSA anti- anti- BSA body/ anti- Doses pri- body/ anti-BSAanti-BSA of adju- mary/ primary antibody/ antibody/ Vaccine vant (in re-re- primary primary compo- mg/ sponse sponse response response sitionsAdjuvant dose) (D14) (D28) (D42) (D56) 1 — — <500 <500 <500 <500 2 GluMn1 64,000 32,000 256,000 256,000 3 GluCa 3 16,000 8000 128,000 96,000 4FruhpCa 1 4000 2000 64,000 48,000 5 GlyCa 2 4000 2000 64,000 64,000 6GluZn 0.5 16,000 6000 96,000 96,000 7 GluhpZn 1 8000 6000 64,000 64,000Control — — <500 <500 <500 <500

The control composition corresponds to mice which were not vaccinated.

The results obtained show that the addition of the adjuvants accordingto the invention to a therapeutic composition induces a significantimmunostimulatory activity as compared with the non-vaccinated controland to the therapeutic compositions not comprising any adjuvants.

It may also be noted that GluMn, although used at a low concentration,allows a particularly high level of antibodies to be obtained.

EXAMPLE 2

The immunostimulatory effect of a water-soluble salt according to theinvention, GluMn was compared with that of an insoluble salt, Al(OH)₃.

The therapeutic compositions (dose/mouse) used, 100 μl in volume,comprised 50 μg of antigen and 1 mg of Al(OH)₃.

Doses not comprising any adjuvants were also injected.

One batch of mice was not vaccinated (control). The results obtained arefeatured in Table III below.

TABLE III IgGl IgGl IgG2_(a) IgG2_(a) IgGl IgGl secon- secon- IgG2IgG2_(a) secon- second- primary primary day dary primary primary darydary response response response response response response responseresponse Adjuvant D14 D28 D42 D56 D14 D28 D42 D56 — <500 <500 <500 <500<500 <500 <500 <500 GluMn 32,000 64,000 256,000 256,000 4000 8000 24,00064,000 Al(OH)₃ 16,000 64,000 128,000 256,000 1000 6000 8000 32,000Control <500 <500 <500 <500 <500 <500 <500 <500

The IgG1s are representative of the humoral response.

The IgG2s are representative of the cell response.

The humoral and cell responses obtained with an adjuvant according tothe invention are greater than those obtained with a conventionaladjuvant such as Al(OH)₃.

EXAMPLE 3

In order to demonstrate the synergy effect between an adjuvant of thewater-soluble salt type with an oily adjuvant (Montanide ISA 25 definedin Table I), the effect of various therapeutic compositions wascompared. Each of them comprised 50 μg of antigen and had a volume of100 μl (dose/mouse).

The results obtained are featured in Table IV below:

TABLE IV Water- Amount of Montanide Humoral Cell soluble salt ISA25immunity immunity salt (mg/dose) (in mg/dose) (D14) (D14) — — 25 24,0003000 GluMn 1 — 64,000 4000 GluMn 1 25 96,000 8000 GluMn 0.5 — 16,000<2000 GluMn 0.5 25 96,000 6000

The combination of an oily adjuvant with a soluble salt according to theinvention makes it possible to obtain an antibody level which issuperior to that obtained by the simple addition of the antibody levelsobtained with each of these adjuvants used individually. Synergy isindeed demonstrated. This synergy is even more pronounced for a lowamount (0.5 mg/dose) of GluMn.

EXAMPLE 4

In order to demonstrate the importance of the organic nature of theanion, the immunostimulatory effect of various calcium salts wascompared.

Each therapeutic composition, 100 μl in volume, contained 50 μg ofantigen and, except for the control, 0.5 mg of a calcium salt, such thatthe calcium concentration of each composition was 2.7 mg.

The antibodies were assayed 42 days after the vaccination.

Injection of the therapeutic composition was repeated on D28. Theantibodies were assayed on D42.

The results obtained are featured in Table V:

TABLE V Salts Humoral response Cell response CaCO₃ 32,000 4000 CaCl₂64,000 24,000 CaHPO₄ 64,000 10,000 GluCa 128,000 32,000 — 3000 <2000

It was observed during these tests that CaCl₂ induced very largelesions.

EXAMPLE 5

A therapeutic composition 100 μl in volume, comprising 50 μg of antigen,was injected into various batches of mice each comprising 5 mice. Thelocal reactions (lesions and granulomae) were evaluated on D8 and D35.

The results obtained are featured in Tables VI and VII respectively.

TABLE VI (D8) Mouse No. Salt concentration (mg/dose) 1 2 3 4 5 GluMn1 + + − − + GlyMn 1 L + ++ + − GluMn⁺ 1 ++ +++ + − + GluK 1 − − − − −Asp K and 1 − − − − − Asp Mg GluCa 1 − − − − − MnCl²⁺ 1 +++ L L L LAl(OH)₃ 1 +++ +++ ++ +++ ++++

TABLE VII (D35) Mouse No. Salt concentration (mg/dose) 1 2 3 4 5 GluMn 1− + + + − GlyMn 1 − + + + + GluMn+ 1 ++ ++ ++ +++ + GluK 1 − − − − − AspK and Asp Mg 1 − − − − − GluCa 1 − − − − − MnCl²⁺ 1 +++ LL LL L LAl(OH)₃ 1 + +++ ++ +++ ++++ (*): GluMn combined with 25 mg of MONTANIDEISA 25 − = no granuloma + = small granuloma ++ = medium granuloma +++ =large granuloma ++++ = very large granuloma − = no lesions L = smalllesion LL = medium lesion LLL = large lesion LLLL = very large lesionDCD = mice that died during the experiment

The chosen concentration of Al(OH)₃ corresponds to the concentration atwhich this salt allows the largest immune response to be obtained. Thisconcentration of Al(OH)₃ is that used in the above examples.

Although only preferred embodiments of the invention are specificallyillustrated and described above, it will be appreciated that manymodifications and variations of the present invention are possible inlight of the above teachings and within the purview of the appendedclaims without departing from the spirit and intended scope of theinvention.

What is claimed is:
 1. An immunogenic composition comprising (i) atleast one antigen or at least one in vivo generator of a compoundcomprising an amino acid sequence and (ii) at least one adjuvantcomprising at least one pharmaceutically acceptable and water-solublesalt of an organic anion and a metal cation, wherein said organic anionis glycerol phosphoric acid, said adjuvant being present in an amounteffective to increase the immune response.
 2. The immunogeniccomposition of claim 1, wherein the metal cation is divalent.
 3. Theimmunogenic composition of claim 2 wherein the metal cation is zinc,manganese or calcium.
 4. The immunogenic composition of claim 3, whereinthe divalent metal cation is manganese.
 5. The immunogenic compositionof claim 1 in an emulsion form comprising at least one aqueous phase andat least one oily phase, the oily phase including said adjuvant.
 6. Theimmunogenic composition of claim 5, wherein the emulsion is of thewater/oil/water, oil/water, or microemulsion type.
 7. The immunogeniccomposition of claim 5, wherein said oily phase including the adjuvantfurther comprises a surface-active agent.
 8. The immunogenic compositionof claim 7, wherein said surface-active agent comprises (i) an esterobtained by condensation of a fatty acid with (a) a sugar, (b) apoly-ethylene glycol, (c) condensation of a fatty acid with (a) a sugar,(b) a poly-ethylene glycol, (c) sorbitol, (d) glycerol, or a derivativeof such an ester whose hydrophilicity has been modified, and (ii) anethoxylated plant oil.
 9. The immunogenic composition of claim 5,wherein the oily adjuvant is a self-emulsifiable oil.
 10. Theimmunogenic composition of claim 9, wherein the oily adjuvant is anester of ethoxylated fatty acids corresponding to one of the followingformulae:

in which R₁, R₃, R₅, R₆, R₈ and R₁₀ represent a saturated orunsaturated, linear or branched hydrocarbon chain having from 5 to 30carbon atoms; R₂, R₄, R₇ and R₉ represent a saturated or unsaturated,linear or branched hydrocarbon chain having from 1 to 5 carbon atoms;the total number of ethylene oxide molecules represented in theabovementioned formulae II, III and IV by k, 1+m and n+p+q,respectively, being an integer such that said compounds have an HLBvalue between about 4 and about 10 (hydrophilic/lipophilic balance). 11.The immunogenic composition of claim 10, wherein said HLB value isbetween about 5 and about
 9. 12. The immunogenic composition of claim 1,further comprising a surface active agent, said composition being in amicellar solution form.
 13. The immunogenic composition of claim 12,wherein the surface-active agent is (i) an ester obtained bycondensation of a fatty acid with a sugar or glycerol, or a derivativeof such an ester whose hydrophilicity has been modified, or (ii) anethoxylated plant oil.
 14. The immunogenic composition of claim 1,wherein said at least one in vivo generator of a compound comprising anamino acid sequence is a recombinant sub-unit vaccine or a recombinantplasmid comprising an exogenous nucleotide sequence which expresses acompound comprising an amino acid sequence in a host organism.
 15. Amethod for treating a functional disease comprising administering to apatient the immunogenic composition of claim
 1. 16. The method of claim15, wherein the salt of said immunogenic composition is combined with(a) an oily adjuvant, (b) a surface-active agent or (c) an oily adjuvantcombined with a surface-active agent.
 17. The method of claim 15 whereinsaid composition further comprises at least one aqueous phase.
 18. Themethod of claim 17 wherein said composition is in an emulsion form. 19.The method of claim 18, wherein said composition is in a micellarsolution form.
 20. A method of making a composition intended for theprevention or treatment of infectious diseases comprising combining (a)at least one adjuvant comprising at least one pharmaceuticallyacceptable and water-soluble salt of an organic anion and a metal cationand (b) at least one antigen or at least one in vivo generator of acompound comprising an amino acid sequence, wherein the organic anion isglycerol phosphoric acid.
 21. The method of claim 20, further comprisingthe step of combining the salt with (a) an oily adjuvant, (b) asurfactant or (c) an oily adjuvant combined with a surface-active agent.22. An immunogenic therapeutic composition comprising (i) at least onecompound capable of eliciting an immune response and (ii) at least oneadjuvant comprising at least one pharmaceutically acceptable andwater-soluble salt of an organic anion and a metal cation, wherein theorganic anion is glycerol phosphoric acid, said adjuvant being presentin an amount effective to increase the immune response.